White blood cells and their secreted products are key elements of immune systems biology and important indicators of patient health and disease. Large numbers of such variables in whole blood and other cell suspensions, including cell populations, cell surface antigens and intracellular molecules may be immunoprofiled using the microvolume laser scanning cytometry (MLSC) system. Immunoprofiling of cells and molecules, and use of a MLSC system are described in Kantor et al., 2004, “Immune Systems Biology: Immunoprofiling of Cells and Molecules,” Biotechniques, 36(3):520-4; in Walton et al., 2000, “Microvolume Laser Scanning Cytometry Platform for Biological Marker Discovery,” Proc. SPIE-Int. Soc. Opt. Eng., 3926:192-201; and in Kantor et al., 2004, “Biomarker Discovery by Comprehensive Phenotyping for Autoimmune Diseases,” Clinical Immunology, 111:186-195, all of which are incorporated herein by reference in their entirety. In addition, the MLSC and related optical cuvette are described in Dietz et al., “System for MicrovolumeLaser Scanning Cytometry,” U.S. Pat. No. 6,687,395 (Feb. 3, 2004); Dietz et al., “Optical architectures for Microvolume Laser-Scanning Cytometers,” U.S. Pat. No. 6,603,537 (Aug. 5, 2003); and Dietz et al., “Disposable Optical Cuvette Cartridge,” U.S. Pat. No. 6,552,784 (Apr. 22, 2003), all incorporated herein by reference in their entirety. The above noted MLSC system of U.S. Pat. No. 6,687,395 incorporates photomultiplier tubes (PMTs) and is optimized for analysis of whole blood, but is limited to three or four color assays because of the limitations of traditional fluorophores and static detection channels of the PMT based detection system.
Prior laser scanning cytometers and flow cytometers depend on the arrangement of dichroic filters to define detection channels with each requiring a dedicated photomultiplier tube. The throughput and sensitivity of these systems is mostly limited by low detector efficiencies (less than 15%), especially in the red region of the spectrum that is used for whole blood assays. Accordingly, there is an unmet need for a polychromatic laser scanning cytometry system with a high level of multiplexing that can be used in clinical studies. Such technology is critical for identifying fine subsets of cells within the extremely complex immune system and relating them to disease pathogenesis.
In MLSC, as in flow cytometry, fluorophore-tagged antibody reagents specific for cellular antigens are used to identify, characterize, and enumerate specific leukocyte populations. In order to operate with whole blood and minimize the effects of auto-fluorescence and light attenuation, fluorophores are used that can be excited in the red and near infrared region (>600 nm) of the spectrum. APC, Cy5, Cy5.5, Cy7APC and some of the Alexa dyes work well in the system. See Mujumdar et al., 1993, “Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters,” Bioconjug Chem, 4(2):105-11; Roederer et al., 1996, “Cy7PE and Cy7APC: Bright New Probes for Immunofluorescence,” Cytometry, 24(3):191-7; Beavis et al., “Allo-7: A New Fluorescent Tandem Dye for Use in Flow Cytometry,”24(4):390-395; Panchuk-Voloshina et al., 1999, “Alexa Dyes, A Series of New Fluorescent Dyes That Yield Exceptionally Bright, Photostable Conjugates,” J. Histochem Cytochem, 47(9):1179-88; and Berlier et al., 2003, “Quantitative Comparison of Long-Wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates,” J. Histochem Cytochem, 51(12):1699-712, all of which are incorporated herein by reference in their entirety.
In contrast to flow cytometry, in a MLSC system the laser scans over stationary cells rather than cells flowing past the laser. A laser beam with a focused spot size appropriate for the imaging of targeted cells and particles is scanned across the sample in one direction while the sample is translated relative to the optical system in a second orthogonal direction. By scanning a predetermined volume for each sample, such as by using a capillary, absolute cell counts (cells per microliter) are obtained directly. In this respect the system is similar to hematology analyzers and different from other laser scanning systems that focus on high throughput screening cell morphology or rare cell applications. See Groner et al., 1995, “Practical Guide to Modern Hematology Analyzers,” Chichester, England: John Wiley and Sons,”; Zuck et al., 1999, “Ligand-receptor Binding Measured by Laser-scanning Imaging,” Proc. Natl. Acad. Sci., 96(20):11122-11127; Martens et al., 1999, “A Generic Particle-based Nonradioactive Homogeneous Multiplex Method for High-throughput Screening Using Microvolume Fluorimetry,” Anal. Biochem, 273(1):20-31; Swartzman et al., 1999, “A Homogeneous and Multiplexed Immunoassay for High-throughput Screening Using Fluorometric Microvolume Assay Technology,” Anal. Biochem., 271(2):143-151; Kamentsky, 2001 “Laser Scanning Cytometry,” Methods Cell. Biol., 63:51-87; and Tibbe et al., 1999, “Optical Tracking and Detection of Immunomagnetically Selected and Aligned Cells,” Nat. Biotechnol., 17(12):1210-1213, all of which are incorporated herein by reference in their entirety. In general, a MLSC system is designed for the performance of many different assays on a large number of patient samples. A comparison between the capabilities of a a MLSC system and flow cytometry is provided in Table 1. Although the scanning times per individual assay are similar, a MLSC system is much more efficient than a flow cytometry system with respect to sample preparation and data analysis. Sample handling and operator interaction are minimized. There is no need to prepare peripheral blood mononuclear cells (PBMC) by density gradients or to wash away unreacted reagent. In terms of processing and absolute cell counts, significant advantages are provided by a MLSC system for whole blood and, to a lesser extent, erythrocyte lysed blood preparations. Informatics tools facilitate automated collection, processing and analysis of the data. This data pipelining approach speeds data flow and reduces user error.
Traditionally, cytometry instruments such as flow-cytometers or MLSC system instruments used photomultiplier tubes (PMT) for the detection of the fluorescence signals. Advantages of PMTs are a large dynamic range and a high bandwidth, allowing for data rates of several hundred kHz. However, PMTs require high voltage power supplies, have low quantum efficiency (especially for the red and infrared) and, because they are basically vacuum tubes, PMTs are inherently mechanically instable. In spite of these significant disadvantages, PMTs have been the fluorescence detector of choice in cytometry for a long time. Typically, the emitted fluorescence is split into a number of separate color channels using dichroic beamsplitters and each channel comes equipped with a dedicated PMT. The number of detection channels is limited by the availability of appropriate dichroic mirrors and is fixed for a given instrument. Due to the optical properties of dichroic beam splitters, the color channels have to have a minimum spectral width, significantly limiting the number of detection channels that can be defined in the range between 650 and 800 nm.
With regard to the general background of CCDs, the basic principle of solid-state CCDs is the photovoltaic effect; that is, photons impacting into a silicon layer create electron-hole pairs. Arrays of surface electrodes are used to keep the charge confined to small rectangular areas called pixels. By manipulating the electrode potentials, the charge can be moved from pixel to pixel towards a charge-sensing node where it ultimately is converted into a digital number using an A/D-converter. CCDs offer the advantages of avoiding high voltage power requirements of PMTs and have higher quantum efficiency than PMT systems. In addition, CCDs are more stable because of their solid-state construction.
There is an ever increasing number of available antibodies to cell surface antigens (e.g., there are more than 200 CD antigens) and intracellular components. In addition, available techniques include analysis of phosphorylated molecules. See Perez et al., 2002, “Simultaneous Measurement of Multiple Active Kinase States Using Polychromatic Flow Cytometry,” Nat Biotechnol, 20(2):155-162; and Perez et al., 2004, “Flow Cytometric Analysis of Kinase Signaling Cascades,” Methods Mol Biol, 263:67-94, both of which are incorporated herein by reference in their entirety. This has driven the highly detailed subsetting of cell populations and inclusion of more and more colors in cytometric analysis. See De Rosa et al., 2003, “Beyond Six Colors: A New Era In Flow Cytometry,” Nat. Med., 9(1):112-117.
Flow cytometry based systems detecting eight or more tags require multiple laser excitations and are not optimized for whole blood applications and absolute cell counting. Multiple tags using multiple laser excitations are described in Roederer et al., 1997, “8 Color, 10-parameter Flow Cytometry to Elucidate Complex Leukocyte Heterogeneity,” Cytometry, 29(4):328-39; Baumgarth et al., 2000, “A Practical Approach to Multicolor Flow Cytometry for Immunophenotyping,” J. Immunol Methods, 243(1-2):77-97; and De Rosa et al., 2001, “11-Color, 13-Parameter Flow Cytometry: Identification of Human Naive T-Cells by Phenotype, Function, and T-cell Receptor Diversity,” Nat. Med., 7(2):245-248, all of which are incorporated herein by reference in their entirety.
The staining reaction for an MLSC sample can be done in whole blood or other single cell suspensions. Peripheral blood mononuclear cells, erythrocyte-lysed blood, synovial fluid, bronchioalveolar lavage, splenocytes, and cell lines have been used in a MLSC system, as have viably frozen and thawed cells. In general, assays can be conducted in homogeneous mode. There is no need to wash the reagent away; quantitative dilution of the blood-antibody mixture is usually sufficient sample preparation. Addition of permeabilization and washing steps enable the monitoring of intracellular antigens. Each capillary array holds 32 separate assays and is compatible with multi-channel pipetting devices. The cell-antibody mixtures are loaded into the capillaries and scanned.
Reporter tags are needed in cellular assays to identify specific components from among the thousands of molecules present in a cell or biological sample. A reporter tag consists of at least two components. The reactive component of the tag is capable of undergoing a highly selective and sensitive reaction with the functional group of interest. Examples of reactive tag components are antibodies specific to the antigen of interest. The second element of the reporter tag is an optically active component, which can absorb energy in the form of photons at one wavelength and release energy in the form of photons with wavelengths different from the excitation wavelength. Examples for optically active components of a reporter tag are organic fluorophores, fluorescent proteins, Quantum Dot crystals and SERS particles. Reporter tags are expected to be thermodynamically stable, compatible with the samples of interest, give a quantitative response proportional to the concentration of the functional group of interest, and must be suitable for multiplexing with other tags. Most suitable for the scanner of the present invention are tags compatible with whole blood samples and optical characteristics that allow for using six or more tags simultaneously with only a single laser source.
As described in by Coons, 1961, “The Beginnings of Immunofluorescence,” J. Immunol, 87:499-503, incorporated herein by reference in its entirety, organic fluorescence labels became popular in the 1950's, with a review of fluorescence labeling of tissue appearing in 1961. Organic dyes, along with fluorescent proteins and tandem dyes are the principal tags used in cytometry today. See Oi et al., 1982, “Fluorescent Phycobiliprotein Conjugates for Analyses of Cells and Molecules,” J. Cell Biol, 93(3):981-6; Glazer et al., 1983, “Fluorescent Tandem Phycobiliprotein Conjugates-Emission Wavelength Shifting by Energy Transfer,” Biophys J, 43(3):383-6; and Waggoner et al., 1993, “PE-CY5—A New Fluorescent Antibody Label for Three-color Flow Cytometry with a Single Laser,” Ann N Y Acad Sci, 677:185-93, all of which are incorporated herein by reference in their entirety. Fluorescence detection is very sensitive, as has been demonstrated by the ability to detect single molecules as described in Ha, 2001, “Single-molecule Fluorescence Resonance Energy Transfer,” Methods, 25(1):78-86 (incorporated herein by reference in its entirety), and signals generated by the tag are proportional to the number of tag molecules interrogated, making it a quantitative technique. Limitations include susceptibility to photobleaching, the limited number of spectrally distinct dyes especially above 600 nm, broad emission spectra, typically 50-100 nm and the small Stoke shift for organic and protein dyes. Thus with a single laser excitation, low levels of multiplexing are possible for organic fluorophores by selecting dyes with different emission wavelengths. Ideally the emission profiles are non-overlapping, as it is difficult to accurately measure a low concentration of one dye in the presence of a large concentration of a second dye, when the profiles overlap. In addition, when dyes are chosen with well-separated emission spectra, the excitation spectra also generally become separated, such that a single excitation wavelength can no longer be used. This can be overcome in part with the preparation of tandem dyes that use energy transfer to extend the effective Stokes shift from excitation to emission. It is apparent however, that simultaneous quantitative detection using organic fluorophores and a single laser excitation is limited to a small number of tags (e.g., 3-5) with emission greater than 600 nm needed for whole blood assays.
In recent years there has been an increase in the use of nanoparticles as biological tags. The driving force behind this development has been the desire to eliminate the use of organic labeling, which have shortcomings, as described above. Much of this work has been in the development of quantum dots (also referred to herein as “Qdots”). Quantum dots take advantage of the quantum confinement effect, giving the nanoparticles unique optical properties. Quantum dots offer advantages over organic dye molecules in that they have brighter emission, significantly narrower emission spectra, and lack the characteristic spectral tail of organic dyes. See Steigerwald et al., 1988 “Surface Derivatization and Isolation of Semiconductor Cluster Molecules,” J. Am. Chem. Soc., 110:3046-3050; and Rosetti et al., 1982, “Electron-Hole Recombination Emission as a Probe of Surface Chemistry in Aqueous Cds Colloids,” J. Phys. Chem., 86:4470-4472, both of which are incorporated herein by reference in their entirety. The average emission spectrum of currently commercially available quantum dots is typically 30-50 nm wide and potentially allows higher levels of multiplexing than with traditional fluorophores. Other key advantages of Qdots for cytometry applications include significantly decreased photobleaching and relatively large Stokes shifts, which enable emissions over a large wavelengths region using a single excitation source. Due to these advantages, quantum dots have begun to be used in a number of real-world biological applications including cellular assays. See Watson et al., 2003, “Lighting up Cells with Quantum Dots,” Biotechniques, 34(2):296-300, 302-3; Wu et al., 2003, “Immunofluorescent Labeling of Cancer Marker Her2 and Other Cellular Targets with Semiconductor Quantum Dots,” Nat Biotechnol, 21(1):41-6; and Bruchez et al., 1998, “Semiconductor Nanocrystals as Fluorescent Biological Labels,” Science, 281(5385):2013-6, all of which are incorporated herein by reference in their entirety. Limitations include the small set of currently available colors, especially in the red and infrared. There are currently two such commercially available tags (Quantum Dot Corp., Hayward, Calif.) with emissions at 655 and 705 nm and it is expected that the set will continue to be expanded. The unique emission lifetime characteristics make Qdots best suited for longer integration times. Over all, simultaneous quantitative detection using Qdots and a single laser excitation has long-term potential for 6 to 10 tags with emission greater than 600, and up to 20 tags with emission greater than 400 nm.
Raman spectroscopy is an optical technique in which the measurement of scattered light is used to identify molecular vibrations and hence elucidate the structure of molecules. While the much more common infrared (IR) spectroscopy also uses the fingerprinting ability of vibrational spectroscopy to identify organic molecules, optical detection in the infrared region does not work well for biological analysis because of the presence of water in biological samples. The extremely dipolar water molecule has strong absorption bands in the IR-region, which would interfere with any signal of the bio-molecule in this region. Raman scattering, however, can be observed in the visible and near-infrared regions, where the absorption of water is much lower. In addition, water by itself is also a weak Raman-scatterer. Thus, even though its concentration in biological samples is orders of magnitudes higher than the molecule of interest, water does not interfere with the Raman signature of the low-concentration tag.
However, Raman scattering is a rare event, and measurements typically have poor sensitivity. Surface enhanced Raman scattering (SERS) was a phenomenon first reported in the 1970's, in which Raman scattering from roughened metal surfaces was found to increase by as much as 106 fold (32, 33). See Van Duyne, 1979, “Laser Excitation of Raman Scattering from Adsorbed Molecules on Electrode Surfaces,” In: Moore C B, editor, Chemical and Biochemical Applications of Lasers, p 101-185; Jeanmaire et al., 1977, “Surface Raman Spectroelectrochemistry Part 1: Heterocyclic, Aromatic, and Aliphatic Amines Adsorbed on the Anodized Silver Electrode,” J. Electroanal. Chem., 84:1-20, both of which are incorporated herein by reference in their entirety. Steady progress was made towards an understanding of the SERS effect through the following decades. See Kneipp et al., 1999, “Ultrasensitive Chemical Analysis by Raman Spectroscopy,” Chem. Rev., 99:2957-2975; and Mulvaney et al., 2000, “Raman Spectroscopy,” Anal. Chem., 145R-157R, both of which are incorporated herein by reference in their entirety. In 1997, Nie and Emory, followed by Kneipp et al. first reported single molecule detection using SERS. See Nie et al., 1997, “Probing Single Molecules and Single nanoparticles by Surface-Enhanced Raman Scattering,” Science, 275:1102-1106; and Kneipp et al., 1997 “Single-Molecule Detection Using Surface-Enhanced Raman Scattering (SERS),” Phys. Rev. Lett., 1667-1670, both of which are incorporated herein by reference in their entirety. Since then numerous reports have verified that SERS is capable of single molecule detection. See Doering et al., 2002, “Single-molecule and Single-nanoparticle Sers: Examining the Roles of Surface Active Sites and Chemical Enhancement,” J. Phys. Chem., 311-317; Constantino et al., 2001, “Single-molecule Detection Using Surface-enhanced Resonance Raman Scattering and Langmuir-blodgett Monolayers,” Anal. Chem., 73:3674-3678; and Bizzarri et al., 2002, “Surface-enhanced Resonance Raman Spectroscopy Signals from Single Myoglobin Molecules,” Appl. Spectrosc, 56:1531-1537, all of which are incorporated herein by reference in their entirety.
Importantly, these measurements indicate an enhancement factor of up to 1014 for single molecules. These results make SERS a candidate technique when measurements with high sensitivity and high multiplexing are required. Of course, single-molecule detection sensitivity is not required for typical cytometry applications. Glass-encapsulated SERS tags were recently reported (44) by Mulvaney et al., 2003, “Glass-coated, Analyte-tagged Nanoparticles: A New Tagging System Based on Detection with Surface-enhanced Raman Scattering,” Langmuir, 19:4784-4790. See also, Natan, “Surface enhanced spectroscopy-active composite nanoparticles” U.S. Pat. No. 6,514,767 (Feb. 4, 2003), incorporated herein by reference in its entirety. Raman scattering is confined to a relative small spectral window around the excitation wavelength. Raman spectra usually are reported using wavenumber units [cm−1]. A typical Raman spectrum extends between 200 cm−1 and 3800 cm−1 from the excitation wavelength, with individual spectral features covering 20 cm−1 to 50 cm−1. Based on these parameters, a Raman spectrum can exhibit up to 150 distinguishable spectral features. Since it is possible to design particles with specific Raman signatures, the number of distinguishable Raman tags excitable with a single laser source greatly exceeds the number of available fluorescent tags. If a Helium-Neon laser is used for excitation, a 20 cm−1-wide Raman-feature corresponds to a spectral bandpass of approximately 1 nm only. Because of these extremely fine spectral features, only instruments with a large number of detection channels or with freely configurable bandpass definition of the detection channels can truly take advantage of the multiplexing capabilities of Raman tags. Both characteristics are implemented in the claimed polychromatic laser-scanning instrument with CCD detection.
Unlike fluorescent tags, Raman tags do not need to gain enough energy to populate an electronically excited state. Thus, photobleaching of the tags is greatly reduced in Raman when compared to fluorophores.